Structural and functional dynamics of an integral membrane protein complex modulated by lipid headgroup charge

We have used membrane surface charge to modulate the structural dynamics of an integral membrane protein, phospholamban (PLB), and thereby its functional inhibition of the sarcoplasmic reticulum Ca-ATPase (SERCA). It was previously shown by electron paramagnetic resonance, in vesicles of neutral lipids, that the PLB cytoplasmic domain is in equilibrium between an ordered T state and a dynamically disordered R state and that phosphorylation of PLB increases the R state and relieves SERCA inhibition, suggesting that R is less inhibitory. Here, we sought to control the T/R equilibrium by an alternative means-varying the lipid headgroup charge, thus perturbing the electrostatic interaction of PLB's cationic cytoplasmic domain with the membrane surface. We resolved the T and R states not only by electron paramagnetic resonance in the absence of SERCA but also by time-resolved fluorescence resonance energy transfer from SERCA to PLB, thus probing directly the SERCA-PLB complex. Compared to neutral lipids, anionic lipids increased both the T population and SERCA inhibition, while cationic lipids had the opposite effects. In contrast to conventional models, decreased inhibition was not accompanied by decreased binding. We conclude that PLB binds to SERCA in two distinct structural states of the cytoplasmic domain: an inhibitory T state that interacts strongly with the membrane surface and a less inhibitory R state that interacts more strongly with the anionic SERCA cytoplasmic domain. Modulating membrane surface charge provides an effective way of investigating the correlation between structural dynamics and function of integral membrane proteins.     

Role of conformational sampling of Ser16 and Thr17-phosphorylated phospholamban in interactions with SERCA

Phosphorylation of phospholamban (PLB) at Ser16 and/ or Thr17 is believed to release its inhibitory effect on sarcoplasmic reticulum calcium ATPase. Ser16 phosphorylation of PLB has been suggested to cause a conformational change that alters the interaction between the enzyme and protein. Using computer simulations, the conformational sampling of Ser16 phosphorylated PLB in implicit membrane environment is compared here with the unphosphorylated PLB system to investigate these conformational changes. The results suggest that conformational changes in the cytoplasmic domain of PLB upon phosphorylation at Ser16 increase the likelihood of unfavorable interactions with SERCA in the E2 state prompting a conformational switch of SERCA from E2 to E1. Phosphorylation of PLB at Thr17 on the other hand does not appear to affect interactions with SERCA significantly suggesting that the mechanism of releasing the inhibitory effect is different between Thr17 phosphorylated and Ser16 phosphorylated PLB.     

Phosphorylation-dependent conformational switch in spin-labeled phospholamban bound to SERCA

We have used chemical synthesis, functional reconstitution, and electron paramagnetic resonance (EPR) to probe the functional dynamics of phospholamban (PLB), which regulates the Ca-ATPase (SERCA) in cardiac sarcoplasmic reticulum. The transmembrane domain of PLB inhibits SERCA at low [Ca(2+)], but the cytoplasmic domain relieves this inhibition upon Ser16 phosphorylation. Monomeric PLB was synthesized with Ala11 replaced by the 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) spin label, which reports peptide backbone dynamics directly. PLB was reconstituted into membranes in the presence or absence of SERCA. TOAC-PLB showed normal inhibitory function, which was reversed by phosphorylation at Ser16 or by micromolar [Ca(2+)]. EPR showed that the PLB cytoplasmic domain exhibits two resolved conformations, a tense T state that is ordered and a relaxed R state that is dynamically disordered and extended. PLB phosphorylation shifts this equilibrium toward the R state and makes it more dynamic (hyperextended). Phosphorylation strongly perturbs the dynamics of SERCA-bound PLB without dissociating the complex, while micromolar [Ca(2+)] has no effect on PLB dynamics. A lipid anchor synthetically attached to the N terminus of PLB permits Ca-dependent SERCA inhibition but prevents the phosphorylation-induced disordering and reversal of inhibition. We conclude that the relief of SERCA inhibition by PLB phosphorylation is due to an order-to-disorder transition in the cytoplasmic domain of PLB, which allows this domain to extend above the membrane surface and induce a structural change in the cytoplasmic domain of SERCA. This mechanism is distinct from the one that relieves PLB-dependent SERCA inhibition upon the addition of micromolar [Ca(2+)].     

Phosphorylation and mutation of phospholamban alter physical interactions with the sarcoplasmic reticulum calcium pump

Phospholamban physically interacts with the sarcoplasmic reticulum calcium pump (SERCA) and regulates contractility of the heart in response to adrenergic stimuli. We studied this interaction using electron microscopy of 2D crystals of SERCA in complex with phospholamban. In earlier studies, phospholamban oligomers were found interspersed between SERCA dimer ribbons and a 3D model was constructed to show interactions with SERCA. In this study, we examined the oligomeric state of phospholamban and the effects of phosphorylation and mutation of phospholamban on the interaction with SERCA in the 2D crystals. On the basis of projection maps from negatively stained and frozen-hydrated crystals, phosphorylation of Ser16 selectively disordered the cytoplasmic domain of wild type phospholamban. This was not the case for a pentameric gain-of-function mutant (Lys27Ala), which retained inhibitory activity and remained ordered in the phosphorylated state. A partial loss-of-function mutation that altered the charge state of phospholamban (Arg14Ala) retained an ordered state, while a complete loss-of-function mutation (Asn34Ala) was also disordered. The functional state of phospholamban was correlated with an order-to-disorder transition of the phospholamban cytoplasmic domain in the 2D co-crystals. Furthermore, co-crystals of the gain-of-function mutant (Lys27Ala) facilitated data collection from frozen-hydrated crystals. An improved projection map was calculated to a resolution of 8 Å, which supports the pentamer as the oligomeric state of phospholamban in the crystals. The 2D co-crystals with SERCA require a functional pentameric form of phospholamban, which physically interacts with SERCA at an accessory site distinct from that used by the phospholamban monomer for the inhibitory association.     

Effect of Phosphorylation on Interactions between Transmembrane Domains of SERCA and Phospholamban

We have used site-directed spin labeling and electron paramagnetic resonance (EPR) to map interactions between the transmembrane (TM) domains of the sarcoplasmic reticulum Ca²⁺-ATPase (SERCA) and phospholamban (PLB) as affected by PLB phosphorylation. In the cardiac sarcoplasmic reticulum, PLB binding to SERCA results in Ca-dependent enzyme inhibition, which is reversed by PLB phosphorylation at Ser16. Previous spectroscopic studies on SERCA-PLB have largely focused on the cytoplasmic domain of PLB, showing that phosphorylation induces a structural shift in this domain relative to SERCA. However, SERCA inhibition is due entirely to TM domain interactions. Therefore, we focus here on PLB’s TM domain, attaching Cys-reactive spin labels at five different positions. In each case, continuous-wave EPR indicated moderate spin-label mobility, with the addition of SERCA revealing two populations, one indistinguishable from PLB alone and another with more restricted rotational mobility, presumably due to SERCA-binding. Phosphorylation had no effect on the rotational mobility of either component but significantly decreased the mole fraction of the restricted component. Solvent-accessibility experiments using power-saturation EPR and saturation-recovery EPR confirmed that these two spectral components were SERCA-bound and unbound PLB and showed that phosphorylation increased the overall lipid accessibility of the TM domain by increasing the fraction of unbound PLB. However—based on these results—at physiological levels of SERCA and PLB, most SERCA would have bound PLB even after phosphorylation. Additionally, no structural shift in the TM domain of SERCA-bound PLB was detected, as there were no significant changes in membrane insertion depth or its accessibility. Therefore, we conclude that under physiological conditions, the phosphorylation of PLB induces little or no change in the interaction of the TM domain with SERCA, so relief of inhibition is predominantly due to the previously observed structural shift in the cytoplasmic domain.     

Probing the interaction of Arg9Cys mutated phospholamban with phospholipid bilayers by solid-state NMR spectroscopy

Phospholamban (PLB) is a 52 amino acid integral membrane protein that interacts with the sarcoplasmic reticulum Ca^2 + ATPase (SERCA) and helps to regulate Ca^2 + flow. PLB inhibits SERCA impairing Ca^2 + translocation. The inhibition can be relieved upon phosphorylation of PLB. The Arg9 to Cys (R9C) mutation is a loss of function mutation with reduced inhibitory potency. The effect R9C PLB has on the membrane surface and the hydrophobic region dynamics was investigated by ³¹P and ²H solid-state NMR spectroscopy in multilamellar vesicles (MLVs). The ³¹P NMR spectra indicate that, like the phosphorylated PLB (P-PLB), the mutated R9C-PLB protein has significantly less interaction with the lipid bilayer headgroup when compared to wild-type PLB (WT-PLB). Similar to P-PLB, R9C-PLB slightly decreases ³¹P T₁ values in the lipid headgroup region. ²H S_CD order parameters of ²H nuclei along the lipid acyl chain decrease less dramatically for R9C-PLB and P-PLB when compared to WT-PLB. The results suggest that R9C-PLB interacts less with the membrane surface and hydrophobic region than WT-PLB. Detachment of the cytoplasmic domain of R9C-PLB from the membrane surface could be related to its loss of function.     

Serine 16 phosphorylation induces an order-to-disorder transition in monomeric phospholamban

Phospholamban (PLB) is a 52 amino acid membrane-endogenous regulator of the sarco(endo)plasmic calcium adenosinetriphosphatase (SERCA) in cardiac muscle. PLB's phosphorylation and dephosphorylation at S16 modulate its regulatory effect on SERCA by an undetermined mechanism. In this paper, we use multidimensional (1)H/(15)N solution NMR methods to establish the structural and dynamics basis for PLB's control of SERCA upon S16 phosphorylation. For our studies, we use a monomeric, fully active mutant of PLB, where C36, C41, and C46 have been mutated to A36, F41, and A46, respectively. Our data show that phosphorylation disrupts the "L-shaped" structure of monomeric PLB, causing significant unwinding of both the cytoplasmic helix (domain Ia) and the short loop (residues 17-21) connecting this domain to the transmembrane helix (domains Ib and II). Concomitant with this conformational transition, we also find pronounced changes in both the pico- to nanosecond and the micro- to millisecond time scale dynamics. The (1)H/(15)N heteronuclear NOE values for residues 1-25 are significantly lower than those of unphosphorylated PLB, with slightly lower NOE values in the transmembrane domain, reflecting less restricted motion throughout the whole protein. These data are supported by the faster spin-lattice relaxation rates (R(1)) present in both the cytoplasmic and loop regions and by the enhanced spin-spin transverse relaxation rates (R(2)) observed in the transmembrane domain. These results demonstrate that while S16 phosphorylation induces a localized structural transition, changes in PLB's backbone dynamics are propagated throughout the protein backbone. We propose that the regulatory mechanism of PLB phosphorylation involves an order-to-disorder transition, resulting in a decrease in the PLB inhibition of SERCA.     

Phospholamban C-terminal Residues Are Critical Determinants of the Structure and Function of the Calcium ATPase Regulatory Complex

To determine the structural and regulatory role of the C-terminal residues of phospholamban (PLB) in the membranes of living cells, we fused fluorescent protein tags to PLB and sarco/endoplasmic reticulum calcium ATPase (SERCA). Alanine substitution of PLB C-terminal residues significantly altered fluorescence resonance energy transfer (FRET) from PLB to PLB and SERCA to PLB, suggesting a change in quaternary conformation of PLB pentamer and SERCA-PLB regulatory complex. Val to Ala substitution at position 49 (V49A) had particularly large effects on PLB pentamer structure and PLB-SERCA regulatory complex conformation, increasing and decreasing probe separation distance, respectively. We also quantified a decrease in oligomerization affinity, an increase in binding affinity of V49A-PLB for SERCA, and a gain of inhibitory function as quantified by calcium-dependent ATPase activity. Notably, deletion of only a few C-terminal residues resulted in significant loss of PLB membrane anchoring and mislocalization to the cytoplasm and nucleus. C-terminal truncations also resulted in progressive loss of PLB-PLB FRET due to a decrease in the apparent affinity of PLB oligomerization. We quantified a similar decrease in the binding affinity of truncated PLB for SERCA and loss of inhibitory potency. However, despite decreased SERCA-PLB binding, intermolecular FRET for Val⁴⁹-stop (V49X) truncation mutant was paradoxically increased as a result of an 11.3-Å decrease in the distance between donor and acceptor fluorophores. We conclude that PLB C-terminal residues are critical for localization, oligomerization, and regulatory function. In particular, the PLB C terminus is an important determinant of the quaternary structure of the SERCA regulatory complex.     

Phospholamban oligomerization, quaternary structure, and sarco(endo)plasmic reticulum calcium ATPase binding measured by fluorescence resonance energy transfer in living cells

Phospholamban (PLB) oligomerization, quaternary structure, and sarco(endo)plasmic reticulum calcium ATPase (SERCA) binding were quantified by fluorescence resonance energy transfer (FRET) in an intact cellular environment. FRET between cyan fluorescent protein-PLB and yellow fluorescent protein-PLB in AAV-293 cells showed hyperbolic dependence on protein concentration, with a maximum efficiency of 45.1 +/- 1.3%. The observed FRET corresponds to a probe separation distance of 58.7 +/- 0.5A(,) according to a computational model of intrapentameric FRET. This is consistent with models of the PLB pentamer in which cytoplasmic domains fan out from the central bundle of transmembrane helices. An I40A mutation of PLB did not alter pentamer conformation but increased the concentration of half-maximal FRET (K(D)) by >4-fold. This is consistent with the previous observation that this putatively monomeric mutant still oligomerizes in intact membranes but forms more dynamic pentamers than wild type PLB. PLB association with SERCA, measured by FRET between cyan fluorescent protein-SERCA and yellow fluorescent protein-PLB, was increased by the I40A mutation without any detectable change in probe separation distance. The data indicate that the regulatory complex conformation is not altered by the I40A mutation. A naturally occurring human mutation (L39Stop) greatly reduced PLB oligomerization and SERCA binding and caused mislocalization of PLB to the cytoplasm and nucleus. Overall, the data suggest that the PLB pentamer adopts a "pinwheel" shape in cell membranes, as opposed to a more compact "bellflower" conformation. I40A mutation decreases oligomerization and increases PLB binding to SERCA. Truncation of the transmembrane domain by L39Stop mutation prevents anchoring of the protein in the membrane, greatly reducing PLB binding to itself or its regulatory target, SERCA.     

Time-resolved FRET reveals the structural mechanism of SERCA–PLB regulation

We have used time-resolved fluorescence resonance energy transfer (TR-FRET) to characterize the interaction between phospholamban (PLB) and the sarcoplasmic reticulum (SR) Ca-ATPase (SERCA) under conditions that relieve SERCA inhibition. Unphosphorylated PLB inhibits SERCA in cardiac SR, but inhibition is relieved by either micromolar Ca²⁺ or PLB phosphorylation. In both cases, it has been proposed that inhibition is relieved by dissociation of the complex. To test this hypothesis, we attached fluorophores to the cytoplasmic domains of SERCA and PLB, and reconstituted them functionally in lipid bilayers. TR-FRET, which permitted simultaneous measurement of SERCA–PLB binding and structure, was measured as a function of PLB phosphorylation and [Ca²⁺]. In all cases, two structural states of the SERCA–PLB complex were resolved, probably corresponding to the previously described T and R structural states of the PLB cytoplasmic domain. Phosphorylation of PLB at S16 completely relieved inhibition, partially dissociated the SERCA–PLB complex, and shifted the T/R equilibrium within the bound complex toward the R state. Since the PLB concentration in cardiac SR is at least 10 times that in our FRET measurements, we calculate that most of SERCA contains bound phosphorylated PLB in cardiac SR, even after complete phosphorylation. 4 μM Ca²⁺ completely relieved inhibition but did not induce a detectable change in SERCA–PLB binding or cytoplasmic domain structure, suggesting a mechanism involving structural changes in SERCA’s transmembrane domain. We conclude that Ca²⁺ and PLB phosphorylation relieve SERCA–PLB inhibition by distinct mechanisms, but both are achieved primarily by structural changes within the SERCA–PLB complex, not by dissociation of that complex.     

Cytoplasmic residues of phospholamban interact with membrane surfaces in the presence of SERCA: A new role for phospholipids in the regulation of cardiac calcium cycling?

The 52-amino acid transmembrane protein phospholamban (PLB) regulates calcium cycling in cardiac cells by forming a complex with the sarco(endo)plasmic reticulum calcium ATPase (SERCA) and reversibly diminishing the rate of calcium uptake by the sarcoplasmic reticulum. The N-terminal cytoplasmic domain of PLB interacts with the cytoplasmic domain of SERCA, but, in the absence of the enzyme, can also associate with the surface of anionic phospholipid membranes. This work investigates whether the cytoplasmic domain of PLB can also associate with membrane surfaces in the presence of SERCA, and whether such interactions could influence the regulation of the enzyme. It is shown using solid-state NMR and isothermal titration calorimetry (ITC) that an N-terminally acetylated peptide representing the first 23 N-terminal amino acids of PLB (PLB_1-23) interacts with membranes composed of zwitterionic phosphatidylcholine (PC) and anionic phosphatidylglycerol (PG) lipids in the absence and presence of SERCA. Functional measurements of SERCA in sarcoplasmic reticulum (SR) vesicles, planar SR membranes and reconstituted into PC/PG membranes indicate that PLB_1-23 lowers the maximal rate of ATP hydrolysis by acting at the cytoplasmic face of the enzyme. A small, but statistically significant, reduction in the inhibitory effect of the peptide is observed for SERCA reconstituted into PC/PG membranes compared to SERCA in membranes of PC alone. It is suggested that interactions between the cytoplasmic domain of PLB and negatively charged phospholipids might play a role in moderating the regulation of SERCA, with implications for cardiac muscle contractility.     

SERCA2a activity correlates with the force-frequency relationship in human myocardium

The present investigation addresses whether protein expression and function of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2a) and phospholamban (PLB) correlate in failing and nonfailing human myocardium. SERCA2a activity and protein expression, PLB phosphorylation, and the force-frequency relationship (FFR) have been determined in right atrium (RA) and left ventricle (LV) from nonfailing (NF, n = 12) and terminally failing [dilated cardiomyopathy (DCM), n = 12] human hearts. Only in LV of DCM hearts was SERCA2a activity significantly decreased [maximal turnover rate (V(max)) = 196 +/- 11 and 396 +/- 30 nmol. mg(-1). min(-1) in LV and RA, respectively], whereas protein expression of SERCA2a in the different chambers was unchanged in NF (3.9 +/- 0.3 and 3.2 +/- 0.4 densitometric units in LV and RA, respectively) and DCM hearts (4.8 +/- 0.8 and 3.4 +/- 0.1 densitometric units in LV and RA, respectively). Phosphorylation of PLB was higher in LV than in RA in NF (Ser(16): 180.5 +/- 19.0 vs. 56.8 +/- 6.0 densitometric units; Thr(17): 174.6 +/- 11.2 vs. 37.4 +/- 8.9 densitometric units) and DCM hearts (Ser(16): 132.0 +/- 5.4 vs. 22.4 +/- 3.5 densitometric units; Thr(17): 131.2 +/- 10.9 vs. 9.2 +/- 2.4 densitometric units). SERCA2a function, but not protein expression, correlated well with the functional parameters of the FFR in DCM and NF human hearts. Regulation of SERCA2a function depends on the phosphorylation of PLB at Ser(16) and Thr(17). However, direct SERCA2a regulation might also be affected by an unknown mechanism.     

Effects of the Arg9Cys and Arg25Cys mutations on phospholamban's conformational equilibrium in membrane bilayers

Approximately, 70% of the Ca²⁺ ion transport into the sarcoplasmic reticulum is catalyzed by the sarcoplasmic reticulum Ca²⁺-ATPase (SERCA), whose activity is endogenously regulated by phospholamban (PLN). PLN comprises a TM inhibitory region and a cytoplasmic regulatory region that harbors a consensus sequence for cAMP-dependent protein kinase (PKA). The inhibitory region binds the ATPase, reducing its apparent Ca²⁺ binding affinity. β-adrenergic stimulation activates PKA, which phosphorylates PLN at Ser 16, reversing its inhibitory function. Mutations and post-translational modifications of PLN may lead to dilated cardiomyopathy (DCM) and heart failure. PLN's cytoplasmic region interconverts between a membrane-associated T state and a membrane-detached R state. The importance of these structural transitions on SERCA regulation is emerging, but the effects of natural occurring mutations and their relevance to the progression of heart disease are unclear. Here we use solid-state NMR spectroscopy to investigate the structural dynamics of two lethal PLN mutations, R9C and R25C, which lead to DCM. We found that the R25C mutant enhances the dynamics of PLN and shifts the conformational equilibrium toward the R state confirmation, whereas the R9C mutant drives the amphipathic cytoplasmic domain toward the membrane-associate state, enriching the T state population. The changes in membrane interactions caused by these mutations may explain the aberrant regulation of SERCA.     

Sites on the cytoplasmic region of phospholamban involved in interaction with the calcium-activated ATPase of the sarcoplasmic reticulum.

Proton NMR studies have shown that when a peptide corresponding to the N-terminal region of phospholamban, PLB(1-20), interacts with the Ca2+ATPase of the sarcoplasmic reticulum, SERCA1a, docking involves the whole length of the peptide. Phosphorylation of Ser16 reduced the affinity of the peptide for the pump by predominantly affecting the interaction with the C-terminal residues of PLB(1-20). In the phosphorylated peptide weakened interaction occurs with residues at the N-terminus of PLB(1-20). PLB(1-20) is shown to interact with a peptide corresponding to residues 378-405 located in the cytoplasmic region of SERCA2a and related isoforms. This interaction involves the C-terminal regions of both peptides and corresponds to that affected by phosphorylation. The data provide direct structural evidence for complex formation involving residues 1-20 of PLB. They also suggest that phospholamban residues 1-20 straddle separate segments of the cytoplasmic domain of SERCA with the N-terminus of PLB associated with a region other than that corresponding to SERCA2a(378-405).     

The alpha-helical propensity of the cytoplasmic domain of phospholamban: a molecular dynamics simulation of the effect of phosphorylation and mutation

We have used molecular dynamics simulations to investigate the effect of phosphorylation and mutation on the cytoplasmic domain of phospholamban (PLB), a 52-residue protein that regulates the calcium pump in cardiac muscle. Simulations were carried out in explicit water systems at 300 K for three peptides spanning the first 25 residues of PLB: wild-type (PLB(1-25)), PLB(1-25) phosphorylated at Ser16 and PLB(1-25) with the R9C mutation, which is known to cause human heart disease. The unphosphorylated peptide maintains a helical conformation from 3 to 15 throughout a 26-ns simulation, in agreement with spectroscopic data. Comparison with simulations of a fourth peptide truncated at Pro21 showed the importance of the region from 17 to 21 in preventing local unfolding of the helix. The results suggest that residues 11-16 are more likely to unfold when specific capping motifs are not present. It is proposed that protein kinase A exploits the intrinsic flexibility of the 11-21 region when binding PLB. In agreement with available CD and NMR data, the simulations show a decrease in the helical content upon phosphorylation. The phosphorylated peptide is characterized by helix spanning residues 3-11, followed by a turn that optimizes the salt-bridge interaction between the side chains of the phosphorylated Ser-16 and Arg-13. Replacing Arg-9 with Cys results in unfolding of the helix from C9 and an overall decrease of the helical conformation. The simulations show that initiation of unfolding is due to increased solvent accessibility of the backbone atoms near the smaller Cys. It is proposed that the loss of inhibitory potency upon Ser-16 phosphorylation or R9C mutation of PLB is due to a similar mechanism, in which the partial unfolding of the cytoplasmic helix of PLB results in a conformation that interacts with the cytoplasmic domain of the calcium pump to relieve its inhibition.     

Solid-state H and N NMR studies of side-chain and backbone dynamics of phospholamban in lipid bilayers: Investigation of the N27A mutation

Phospholamban (PLB) is an integral membrane protein regulating Ca²⁺ transport through inhibitory interaction with sarco(endo)plasmic reticulum calcium ATPase (SERCA). The Asn27 to Ala (N27A) mutation of PLB has been shown to function as a superinhibitor of the affinity of SERCA for Ca²⁺ and of cardiac contractility in vivo. The effects of this N27A mutation on the side-chain and backbone dynamics of PLB were investigated with ²H and ¹⁵N solid-state NMR spectroscopy in phospholipid multilamellar vesicles (MLVs). ²H and ¹⁵N NMR spectra indicate that the N27A mutation does not significantly change the side-chain or backbone dynamics of the transmembrane and cytoplasmic domains when compared to wild-type PLB. However, dynamic changes are observed for the hinge region, in which greater mobility is observed for the CD₃-labeled Ala24 N27A-PLB. The increased dynamics in the hinge region of PLB upon N27A mutation may allow the cytoplasmic helix to more easily interact with the Ca²⁺-ATPase; thus, showing increased inhibition of Ca²⁺-ATPase.     

Electron paramagnetic resonance reveals a large-scale conformational change in the cytoplasmic domain of phospholamban upon binding to the sarcoplasmic reticulum Ca-ATPase

We used EPR spectroscopy to probe directly the interaction between phospholamban (PLB) and its regulatory target, the sarcoplasmic reticulum Ca-ATPase (SERCA). Synthetic monomeric PLB was prepared with a single cytoplasmic cysteine at residue 11, which was then spin labeled. PLB was reconstituted into membranes in the presence or absence of SERCA, and spin label mobility and accessibility were measured. The spin label was quite rotationally mobile in the absence of SERCA, but became more restricted in the presence of SERCA. SERCA also decreased the dependence of spin label mobility on PLB concentration in the membrane, indicating that SERCA reduces PLB-PLB interactions. The spin label MTSSL, attached to Cys11 on PLB by a disulfide bond, was stable at position 11 in the absence of SERCA. In the presence of SERCA, the spin label was released and a covalent bond was formed between PLB and SERCA, indicating direct interaction of one or more SERCA cysteine residues with Cys11 on PLB. The accessibility of the PLB-bound spin label IPSL to paramagnetic agents, localized in different phases of the membrane, indicates that SERCA greatly reduces the level of interaction of the spin label with the membrane surface. We propose that the cytoplasmic domain of PLB associates with the lipid surface, and that association with SERCA induces a major conformational change in PLB in which the cytoplasmic domain is drawn away from the lipid surface by SERCA.     

Pbp1 Is Involved in Ccr4- and Khd1-Mediated Regulation of Cell Growth through Association with Ribosomal Proteins Rpl12a and Rpl12b

The Saccharomyces cerevisiae Pbp1 [poly(A)-binding protein (Pab1)-binding protein] is believed to be involved in RNA metabolism and regulation of translation, since Pbp1 regulates a length of poly(A) tail and is involved in stress granule (SG) formation. However, a physiological function of Pbp1 remains unclear, since the pbp1Δ mutation has no obvious effect on cell growth. In this study, we showed that PBP1 genetically interacts with CCR4 and KHD1, which encode a cytoplasmic deadenylase and an RNA-binding protein, respectively. Ccr4 and Khd1 modulate a signal from Rho1 in the cell wall integrity pathway by regulating the expression of RhoGEF and RhoGAP, and the double deletion of CCR4 and KHD1 confers a severe growth defect displaying cell lysis. We found that the pbp1Δ mutation suppressed the growth defect caused by the ccr4Δ khd1Δ mutation. The pbp1Δ mutation also suppressed the growth defect caused by double deletion of POP2, encoding another cytoplasmic deadenylase, and KHD1. Deletion of the gene encoding previously known Pbp1-interacting factor Lsm12, Pbp4, or Mkt1 did not suppress the growth defect of the ccr4Δ khd1Δ mutant, suggesting that Pbp1 acts independently of these factors in this process. We then screened novel Pbp1-interacting factors and found that Pbp1 interacts with ribosomal proteins Rpl12a and Rpl12b. Similarly to the pbp1Δ mutation, the rpl12aΔ and rpl12bΔ mutations also suppressed the growth defect caused by the ccr4Δ khd1Δ mutation. Our results suggest that Pbp1 is involved in the Ccr4- and Khd1-mediated regulation of cell growth through the association with Rpl12a and Rpl12b.     

Protein-Protein Interactions in Calcium Transport Regulation Probed by Saturation Transfer Electron Paramagnetic Resonance

We have used electron paramagnetic resonance (EPR) to probe the homo- and heterooligomeric interactions of reconstituted sarcoplasmic reticulum Ca-ATPase (SERCA) and its regulator phospholamban (PLB). SERCA is responsible for restoring calcium to the sarcoplasmic reticulum to allow muscle relaxation, whereas PLB inhibits cardiac SERCA unless phosphorylated at Ser¹⁶. To determine whether changes in protein association play essential roles in regulation, we detected the microsecond rotational diffusion of both proteins using saturation transfer EPR. Peptide synthesis was used to create a fully functional and monomeric PLB mutant with a spin label rigidly coupled to the backbone of the transmembrane helix, while SERCA was reacted with a Cys-specific spin label. Saturation transfer EPR revealed that sufficiently high lipid/protein ratios minimized self-association for both proteins. Under these dilute conditions, labeled PLB was substantially immobilized after co-reconstitution with unlabeled SERCA, reflecting their association to form the regulatory complex. Ser¹⁶ phosphorylation slightly increased this immobilization. Complementary measurements with labeled SERCA showed no change in mobility after co-reconstitution with unlabeled PLB, regardless of its phosphorylation state. We conclude that phosphorylating monomeric PLB can relieve SERCA inhibition without changes in the oligomeric states of these proteins, indicating a structural rearrangement within the heterodimeric regulatory complex.